PROJECT SUMMARY Chlamydia is the most commonly reported sexually transmitted infection of bacterial origin in the US. Infections are often asymptomatic and, if left untreated, can lead to pelvic inflammatory disease, ectopic pregnancy, and infertility. The causative agent is the obligate intracellular pathogen Chlamydia trachomatis, which, upon infec- tion of epithelial cells of the genital tract, establishes its replication niche within a membrane-bound compart- ment called the inclusion. Central to inclusion maturation is a subset of type III translocated effector proteins, the Inc proteins, which by their strategic insertion into the inclusion membrane can intersect the vesicular and non-vesicular trafficking pathways of the host. Our lab has shown that two of these Inc proteins, IncD and IncV, are enriched at ER-inclusion membrane contact sites (MCS), which are area where the endoplasmic reticulum (ER) and the inclusion membrane are maintained in close apposition. In uninfected cells, the ER establishes MCS with various organelles and MCS biology (i.e. non vesicular transfer of lipid or ion) relies on functional components involved in lipid or ion transfer and structural components that tether the organelles in close appo- sition. However, there is increasing recognition that functional components can also act as tethering molecules. Here, we aim to determine if this concept extends to MCS in the context of Chlamydia infection by investigating the mechanisms by which the ER and the inclusion membranes are tethered to one another. We built upon our previous results showing that IncV, through direct interaction with the ER-resident protein VAP and by molecu- lar mimicry of eukaryotic VAP binding motifs (i.e. FFAT motifs), is sufficient to tether the ER to the inclusion. However, our genetic data indicate that, although sufficient, IncV is not essential for MCS formation, suggest- ing that redundant mechanisms exist. Interestingly, our group has previously shown that at ER-Inclusion MCS, the bacterial Inc protein IncD interacts with the host ceramide transfer protein CERT, which binds to the ER- resident VAP proteins. The IncD/CERT/VAP complex is considered a functional component because of its pro- posed role in lipid transfer. However, a potential role for the IncD/CERT/VAP complex in tethering has not been investigated. In this proposal will test the central hypothesis that ER-inclusion MCS formation relies on the re- cruitment of the ER-resident VAP protein through interaction with the Chlamydia Inc proteins IncV and IncD. In Aim1, we will determine the mechanism supporting the IncV-dependent formation of MCS. In Aim2, we will de- termine the respective contributions of the IncV-VAP and IncD-CERT-VAP complexes in ER-Inclusion MCS formation. Altogether our approach will lead to a better understanding of the mechanisms used by Chlamydia to tether the ER to the inclusion, which may reveal novel therapeutic targets to treat and prevent Chlamydia infections. !